The Human Immunodeficiency Virus (HIV) trans-activator of transcription (Tat) is a variable RNA binding peptide of 86 to 110 amino acids in length that is encoded on two separate exons of the HIV genome. Tat is highly conserved among all human lentiviruses and is essential for viral replication. When lentivirus Tat binds to the TAR (trans-activation responsive) RNA region, transcription (conversion of viral RNA to DNA then to messenger RNA) levels increase significantly. It has been demonstrated that Tat increases viral RNA transcription and it has been proposed that Tat may initiate apoptosis (programmed cell death) in T4 cells and macrophages (a key part of the body's immune surveillance system for HIV infection) and possibly stimulates the over production of alpha interferon (α-interferon is a well established immunosuppressive cytokine).
Extracellular Tat's presence early in the course of HIV infection could reduce a patient's immune response, giving the virus an advantage over the host. Furthermore, the direct destruction of T4 cells and induction of α-interferon production could help explain the lack of a robust cellular immune response seen in Acquired Immunodeficiency Syndrome (AIDS) patients, as well as accounting for the initial profound immunosuppression.
However, Tat protein isolated from HIV-infected long term non-progressors (LTNP) is different from C-Tat found in AIDS patents. The Tat protein found in LTNP is capable of trans-activating viral RNA, however, LTNP Tat (designated herein after as IS-Tat for immunostimulatory Tat) does not induce apoptosis in T4 cells or macrophages and is not immunosuppressive. Moreover, T4 cells infected ex vivo with HIV isolated from LTNP (such cell lines are designated Tat TcL) can result in the over expression of IS-Tat proteins, often to the virtual exclusion of other viral proteins, that are strongly growth promoting rather than pro-apoptotic. The Tat genes cloned from these Tat TcLs reveal sequence variations in two Tat regions, at the amino terminus and within the first part of the second exon. These surprising discoveries could help explain why HIV infected LTNP T4 cells do not die off at the staggering rate seen in HIV infected individuals that progress to AIDS.
Additionally, variants of Tat are found in lentiviruses which infect monkey species yet do not result in the development of immunodeficiency and epidemic infection. These variant Tat proteins direct monocyte differentiation into dendritic cells (DCs) which stimulate cytotoxic T lymphocyte (CTL) responses. These simian Tat variants, and other Tat variants that are not immunosuppressive, have been termed attenuated or immunostimulatory Tat (IS-Tat).
Based on observations with long-term CD4+ Tat T cell lines, clinical observations, and experiments in animals, attenuated Tat (more specifically IS-Tat or, alternatively, Tat proteins that have been chemically or physically altered) may act as an immune stimulant activating T4 cells inducing their proliferation. This principle may help to explain the stable T4 levels seen in LTNP. Moreover, attenuated Tat may be useful as an adjuvant when co-administered with other active vaccine components such as, but not limited to, vaccines for other viruses, bacteria, rickettsia and cancer cells.
Cancers and chronic infections are the most prominent examples of common human diseases that respond to immune-based treatments. Although infections were the first diseases to be controlled by immunization, clinical trials in humans have established that an immune response, particularly of the CTL arm of the immune system, could regress some human melanomas and renal cancers. These observations were broadened by the discovery that DCs, a specific class of antigen-presenting cells (APC), are particularly effective at initiating CTL activity against cancers and other diseases. Technologies that target and activate DC have yielded some early successes against human cervical pre-malignancies, caused by infection with Human Papilloma Virus (HPV) and human lung cancer. In contrast to chemotherapeutic drugs currently used against cancer, agents that provoke a CTL response against cancer potentially are accompanied by few side effects, owing to the great specificity of the immune response.
Efforts to develop immunotherapeutic drugs that treat cancer have been hampered by technical difficulties in targeting and activating DC to deliver and sustain the required entry signals to the CTL. Antigen targeting for the induction of a CTL response is a challenge insofar as natural processing requires that the antigen enter the cytoplasm of the cell in order to bind to the immune system's major histocompatibility complex (MHC) Class I antigen, a prerequisite to CTL activation because the ligand for activating the T cell receptor on CTL is a complex of antigen and MHC Class I. In almost all cases, protein antigens, even when they are coupled with a DC co-activator, enter exclusively into the alternative MHC Class II antigen presentation pathway that excludes CTL stimulation. This can be overcome, in part, by peptide-based technologies, because peptides bind to MHC Class I that is already on the surface of the DC. However, this technology is non-specific and most peptides are poor DC activators, which limits their efficacy as human treatments for cancer.
A limited group of biological proteins are known to stimulate a CTL response. Variants and derivatives of the Human Immunodeficiency Virus 1 (HIV-1) trans-activator of transcription (Tat) can stimulate this CTL response. Additional biologics that are currently known to directly trigger a CTL response are based on heat shock proteins (HSP), or on the outer coat protein of certain bacteria. Heat shock proteins have shown limited efficacy in the treatment of certain genital neoplasms related to HPV infection.
Breast cancer is a leading cause of cancer-related deaths in women worldwide. Approximately 1 million new breast cancer cases occur annually resulting in 370,000 deaths around the world. More than 200,000 new cases of invasive breast cancer are diagnosed in the US each year, with approximately 45,000 deaths attributed to this disease making breast cancer the second leading cause of cancer mortality in the U.S. among women, and the fifth leading cause of cancer deaths overall. After a steady decline in morbidity from breast cancer, mean breast cancer survival from time of diagnosis of widely-invasive (Stage 4) disease has not changed over the last two decades. The five year survival rate for stage 4 breast cancer has remained at about 20% since 1988 meaning that the survival advantages of the newer agents have run their course by end stage disease.
Treatment of breast cancer in the adjuvant setting experienced significant improvements over the last forty years. In addition to better tumorectomy, radiotherapy, standard chemotherapy and hormone replacement therapy, new classes of therapies emerged with distinct oncolytic mechanisms, such as TAXOL® (paclitaxel) and HERCEPTIN® (trastuzumab). HERCEPTIN® was the last of these agents to be introduced in 2003. It has not significantly expanded in patient reach since 2007. Additionally HERCEPTIN® efficacy is limited to only 20% of women with breast cancer, those who overexpress the Her2/neu oncogene most prominently. Thus, new and more obliterative agents are needed to combat and prevent breast cancer.
Under investigation to improve management of many cancers, immunotherapy is one targeted mechanism that could control tumor growth and prevent metastases while avoiding many of the side effects associated with standard therapies. This latter consideration is particularly important insofar as breast cancer is a disease that disproportionately affects younger women of childbearing age. Early breast cancer immunotherapy research focused on ways to target the natural immune response against cancer cells by administering either a vaccine or monoclonal antibody for a breast cancer antigen. While this approach made good sense owing to breast cancer being a rich source of tumor-specific proteins (e.g. the lactation-antigens mammaglobin A and lactadherin among others) it proved largely unsuccessful because antibody, in contrast to cytolytic T cell activation, appears to have limited utility for controlling solid tumor growth under most settings.
Next generation breast cancer immunotherapies have focused on ways to enhance the pre-existing anti-breast cancer immune response of the patient based on the theory that immune suppression also limited the efficacy of tumor-targeting strategies. One such immunotherapeutic is a monoclonal antibody directed against CTLA4, a receptor on cytolytic T cells implicated in suppression. While demonstrating some promise against melanoma and ovarian cancer, anti-CTLA4 has proven ineffective as a stand-alone agent in animal models of breast cancer including those employed in the studies reported here. A second class of immunostimulants evaluated in cancers, the toll-like receptor (TLR) agonists, work by initiating new triggering signals into the immune system from monocyte-derived dendritic cells. These agents to date have demonstrated limited utility in most solid cancers including breast cancer, in part because they rapidly induce immunosuppression concomitant to T cell activation.
Human Immunodeficiency Virus infection initiates a progressive immunosuppression that, absent treatment, routinely progresses to AIDS and thereafter death of the infected individual. As immunosuppression is implicated in various models of solid cancer progression, including breast cancer, it is not surprising that HIV-infected persons are at increased risk for various malignancies, specifically non-Hodgkin lymphoma (NHL), Kaposi sarcoma (KS) and invasive cervical cancer, which are AIDS-defining cancers in HIV-infected individuals. Paradoxically, at least three groups have reported a decreased risk for invasive breast cancer in women with progressive HIV disease. HIV-infected women have a statistically significant pattern of decreasing relative risk (RR) for breast cancer when compared with the French general population. Following an AIDS epidemic in Tanzania, a second group found a statistically significant decrease in the incidence of breast cancer, in both men and women. Thirdly, a US consortium analyzing over 8500 cases of progressive HIV disease reported a statistically significant decreased risk (p<0.05) in the development of breast cancer that reverted to baseline once control of viral replication was achieved.